Cancer is one of the diseases with highest incidence in the world. WHO data shows that just in 2008, the number of people who died of cancer reaches up to 760 million, in which 60% come from low-income or middle-income countries and the number will increasingly grow in future. Among more than 200 kinds of cancers, patients with breast cancer, lung cancer, intestinal cancer and pancreas cancer account for 54% of all newly increased cases. At present, the methods for the treatment of cancers mainly comprise surgery, radiotherapy, chemotherapy and the combination thereof according to the types and stages of cancers. There are a number of chemotherapeutic drugs used today. The most common chemotherapy agents act by killing fast-dividing cells, one of the main properties of most cancer cells. Therefore, such drugs can also kill other normal rapidly-dividing cells, such as cells in bone marrow, digestive tract and hair follicles while killing rapidly-dividing cancer cells, and thus generate serious side effect and damage normal tissues. Hence there is a need to develop a new therapeutic drug with small toxic and side effect and hight antitumor efficacy. The dominant strategy for the development of new antitumor drugs is to improve the tumor selectivity of a drug to tumor and to reduce the distribution thereof in normal tissues.
Bufalin (3β,14-dihydroxy-5β, 20(22)-bufadienolide, 5β,20(22)-bufadienolide-3β,14-diol), a main antitumor ingredient in Venenum Bufonis that is a kind of traditional Chinese medicine, is white serous fluid secreted from the parotid gland of Bufo bufo gargarizans or Bufo melanostictus, and can be extracted from Venenum Bufonis. Also, it can be artificially synthesized according to U.S. Pat. Nos. 3,134,772 and 3,687,944. Bufalin is digoxin-like immunoreactive ingredient, and shows various bioactivities such as heart strengthening, anesthetization and vascular stimulation. Since the discovery of the antitumor effect of bufalin in 1994 (Numazawa S, et al. J Cell Physiol, 1994, 160(1): 113-20), a large number of studies have been made, which have found that it has broad-spectrum antitumor effect. Additionally, bufalin exhibits chemosensitization effect on tumor cells in combination with other chemotherapeutic drugs such as Sorafenib and the like (Gao Y, et al. Mol Biol Rep. 2012, 39(2):1683-9). In recent years, there have been more researches that have found bufalin can induce cell apoptosis so as to inhibit the proliferation of various cancer cells such as cells of liver cancer, bone tumor, colon cancer, lung cancer, pancreas cancer, ovarian cancer, gastric cancer, prostate cancer and leukemia (Han K Q, et al. World J Gastroenterol. 2007, 13(24):3374-9; Amano Y, et al. J Steroid Biochem Mol Biol. 2009, 114(3-5):144-51; Li D, et al. Anticancer Drugs. 2009, 20(1):59-64; Yu C H, et al. Cancer Sci. 2008, 99(12):2467-76; Takai N, et al. Int J Mol Med. 2008, 21(5):637-43; Yeh J Y, et al. Prostate. 2003, 54(2):112-24; Yin J Q, et al. Acta Pharmacol Sin. 2007, 28(5):712-20; Zhu Z et al. Int. J. Mol. Sci. Int J Mol Sci. 2012, 13(2):2025-35; Xie C M, et al. Free Radic Biol Med. 2011, 51(7):1365-75). Bufalin can trigger cancer cell apoptosis by activating cell death receptor and mitochondrion pathway (Sun L, et al. Evid Based Complement Alternat Med. Epub 2011 Jun. 18). These studies hint that bufalin can be used as a chemotherapeutic drug for the treatment of cancers. However, due to high toxicity, poor water solubility, short half-life, narrow therapeutic window and the toxic dosage and therapeutic dose being close, the clinical application of bufalin is severely restricted (Gong L L et al. Food and Drug. 2007, 9(10):51-3). Moreover, since it is widely distributed in vivo, other clinical side effects, such as vascular stimulation, anaphylactic shock, hyperpyrexia, sinus bradycardia, etc., are further induced (Dasgupta A, et al. Life Sci. 1998, 63(9):781-8; Bick R J, et al. Life Sci. 2002, 72(6):699-709; Kostakis C, Byard R W. Forensic Sci Int. 2009, 188:e1-e5).
At present, a Huachasu injection, prepared by dissolving Venenum Bufonis in normal saline, has been used for the clinical treatment of cancer in China. It has been reported that the combination of gemcitabine-oxaliplatin with Huachasu can enhance the chemotherapeutic effect in the treatment of patients with advanced gallbladder cancer (Qin T J, et al. World J Gastroenterol. 2008, 14(33):5210-6). Other studies show that the Huachasu injection did not produced obvious toxicity when administered to a patient with liver cancer or pancreas cancer in 8-fold standard dose (20 ml/m2/day or 20-25 ml/person/day, containing 14.3±0.03 ng/ml of bufalin) (Meng Z. et al. Cancer, 2009, 115(22):5309-18), which means that the effective therapeutic amount of bufalin tolerated by an adult patient every day can reach up to 2.3 μg. However, the Huachasu injection is a mixture solution of alkaloids in Venenum Bufonis, and the content of bufalin contained therein is very low due to the poor water solubility of bufalin.
Therefore, it is very necessary to develop a new dosage form which can prolong the duration of bufalin in tumor focus, improve tumor targeting and reduce toxic and side effect.
Liposome has a closed phospholipid dilayer structure with internal water phase. We adopt liposome as the drug carrier of bufalin to solve the problem of poor water solubility thereof by loading bufalin with phospholipid membrane. Additionally, the PEG-modified liposome can allow bufalin to passively target to tumor site so as to reduce the toxic and side effects thereof.
In the last 20 years, liposome was widely used for encapsulating antitumor agents. There have been various antitumor liposome drugs used or to be used clinically, such as doxorubicin liposome (Doxil/Caelyx is sold by Alza/Johnson and Johnson in America or by Schering-Plough in other countries respectively), daunorubicin liposome (DaunoXome, Gilead), cytarabine liposome (DepoCyte, produced by Skye Pharma/Enzon/Mundipharma) and cis-platinum liposome (Lipoplatin, produced by Regulon), wherein the cytarabine liposome can be used for the treatment of meningeal lymphoma. The liposome can improve the solubility of an anti-cancer drug with poor water solubility through phospholipid membrane structure, and realize the passive tumor targeting by PEG modification and the active tumor targeting by conjugating with a carrier.
Conventional liposome is formed by dispersing phospholipid in water phase, thus amphipathic and lipid-soluble agents can be inserted into the phospholipid membrane structure of liposome, whereas hydrophilic agents are directly encapsulated into the internal water phase of liposome. The liposome carrier has a great influence on the pharmacokinetics, distribution in tissue, and toxic and side effect of the encapsulated agent. For example, clinical tests prove that the toxic effect of doxorubicin liposome is significantly reduced as compared with that of doxorubicin monomer while maintaining antitumor activity (Safra T. Oncologist. 2003, 8 Suppl 2:17-24).
However, since the liposome can be rapidly captured by macrophage, its retention time in vivo after administration is only several hours. In order to improve the circulating half-life of liposome in vivo, glycolipids or hydrophilic polymers such as PEG can be applied to liposome. PEG, a biocompatible polymer, is conjugated onto the liposome to enable it to have a protective hydrophilic surface, and the liposome thus obtained is known as “second-generation liposome” or “stealth liposome”. PEG results in steric hindance effect on the surface of liposome so as to hinder the adsorption of opsonins and plasma proteins and reduce the binding of macrophage receptors to phosphate groups on the phospholipid membrane, and thereby prolongs the retention time thereof in blood circulation.
Furthermore, the pharmacokinetic property of liposome can be changed by chemically modifying the surface of liposome with phospholipid groups or binding proteins, polypeptides or other macromolecules. PEG liposomes with conjugated carriers such as micromolecules, peptides or monoclonal antibodies have been widely used in the tumor treatment, e.g. folic acid-conjugated daunorubicin and doxorubicin liposomes (Pan X Q, Lee R J. Anticancer Res. 2005, 25(1A):343-6; Shmeeda H, et al. Mol Cancer Ther. 2006, 5(4):818-24), laminin liposome (Zalipsky S, et al. Bioconjug Chem. 1995, 6(6):705-8) or OV-TL3 monoclonal antibody-conjugated liposome (Vingerhoeds M H, et al. Br J Cancer. 1996, 74(7):1023-9).
So far, there hasn't been any report about bufalin liposome. For this purpose, the present invention is provided.